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Department of biochemistry
3
Selected Lettuce varieties for biological evaluation
6
Research Objectives
7
Enzyme Inhibition Assays
8
α-glucosidase inhibition assay
• Digest carbohydrates
• Facilitates absorption of glucose from intestine
α-glucosidase
9
(Matsui et al., 2007)
Cont…
Sr. No. Sample Percentage inhibition IC50 µg/ml
1000 µg/ml 500 µg/ml 250 µg/ml
1 A2 55.0±2.6 20.2±2.8 4.0±0.6 914.9
2 E2 34.4±4.0 13.3±2.8 2.7±0.6 1405
3 A3 38.3±1.1 15.1±1.3 2.2±1.1 1268
4 E3 71.3±4.1 29.7±2.6 5.9±0.6 701.6
5 A4 12.7±1.5 5.5±0.6 1.2±0.1 3654
6 E4 64.2±1.8 26.9±0.5 5.5±0.1 776.0
7 A6 18.3±1.7 6.6±1.4 1.0±0.7 2288
8 E6 66.7±3.9 23.1±5.9 4.9±1.6 775.3
9 A7 31.1±4.8 15.8±1.9 3.0±0.6 1658
10 E7 58.4±4.6 25.4±1.9 5.2±0.3 847.4
11 A8 27.5±1.3 12.3±1.2 2.4±0.1 1805
12 E8 28.4±4.7 11.4±2.1 2.3±0.4 1687
13 A9 26.0±3.1 10.9±1.3 2.3±0.2 1865
14 E9 74.8±1.7 32.4±1.2 6.4±0.1 662.4
15 A10 60.3±0.6 23.4±2.7 4.3±0.9 835.4
16 E10 72.8±1.5 30.7±0.6 6.3±0.1 685.0
17 A11 41.3±4.3 17.2±2.3 3.7±0.5 1200
18 E11 33.2±2.0 13.5±1.7 2.9±0.1 1472
19 A12 51.8±3.8 22.3±1.5 4.4±0.4 957.5
20 A13 25.8±4.8 9.8±2.8 2.1±0.4 1806
21 E13 49.5±2.5 21.6±0.9 4.4±0.1 1002
10
α-amylase inhibition assay
+ive controls
-ive control
11
(Keerthana et al., 2013)
Cont…
Sr. No. Sample Percentage inhibition IC50 µg/ml
1000 µg/ml 500 µg/ml 250 µg/ml
1 A2 15.2±2.2 5.9±1.0 2.3±0.3 3161
2 E2 25.7±4.0 12.3±1.9 6.0±0.8 2337
3 A3 12.1±2.0 5.8±0.8 2.3±1.2 5091
4 E3 28.4±2.2 13.7±1.0 6.4±5.0 2050
5 A4 10.4±0.4 4.8±0.1 2.1±2.4 5923
6 E4 24.3±0.3 11.3±0.1 5.4±4.3 2427
7 A6 9.8±0.9 4.3±0.2 2.2±2.2 6354
8 E6 31.2±2.1 17.1±1.2 8.3±0.9 1982
9 A7 7.9±1.7 2.4±0.9 1.1±1.2 4297
10 E7 35.5±1.9 16.4±0.7 9.1±3.1 1585
11 A8 10.7±2.2 5.1±1.1 2.5±0.2 6517
12 E8 23.0±2.1 10.1±1.5 4.8±1.0 2464
13 A9 9.0±1.0 3.8±0.6 1.9±2.1 6377
14 E9 30.5±0.7 14.7±0.2 7.2±4.2 1900
15 A10 9.3±0.2 3.9±0.1 1.8±1.1 5838
16 E10 29.0±2.7 16.7±1.3 6.6±3.2 2124
17 A11 9.6±0.3 4.0±0.1 1.5±0.4 5126
18 E11 38.0±2.6 16.9±1.4 7.5±1.4 1386
19 A12 8.0±0.6 3.5±0.2 1.3±0.2 6485
20 A13 8.4±0.4 4.1±0.1 2.1±0.7 9315
21 E13 32.1±5.1 16.3±2.9 8.3±3.1 1796
12
Butrylcholinesterase inhibition assay
-ive control
13
(Jabeen et al., 2014)
Butrylcholinesterase assay
-ive control
15
Cont…
Sr. No. Sample Percentage inhibition IC50 µg/ml
5mg/ml 2.5mg/ml 1.25mg/ml
1 A2 93.4±0.0 44.2±1.2 19.2±0.2 500.7
2 E2 82.0±3.0 39.1±3.9 11.7±0.2 578.6
3 A3 62.4±3.3 25.6±3.1 10.7±0.3 796.8
4 E3 100±0.0 47.2±1.1 18.7±2.1 480.4
5 A4 91.3±2.2 40.3±2.1 15.4±2.0 537.5
6 E4 90.3±2.9 35.8±1.1 9.9±0.3 574.2
7 A6 93.1±3.3 41.3±2.1 20.1±1.1 514.3
8 E6 100±0.0 49.4±1.1 19.9±1.1 464.7
9 A7 77.2±0.8 33.2±1.4 12.2±0.4 634.4
10 E7 100±0.0 47.1±1.4 22.3±0.3 466.7
11 A8 63.4±2.5 32.3±1.2 10.3±0.4 746.1
12 E8 100±0.0 50.1±2.1 28.6±0.2 424.9
13 A9 100±0.0 45.4±1.1 14.2±1.0 504.5
14 E9 100±0.0 48.6±1.1 23.3±1.1 455.0
15 A10 100±0.0 47.2±4.3 20.4±1.9 473.9
16 E10 70.7±4.6 32.4±2.1 14.4±2.2 674.3
17 A11 70.6±3.9 31.3±2.1 15.3±1.1 680.0
18 E11 100±0.0 49.0±2.1 20.1±1.9 465.9
19 A12 52.8±0.7 18.3±1.2 4.2±2.1 952.7
20 A13 98.2±1.8 39.9±2.7 15.8±2.1 531.7
21 E13 100±0.0 50.4±2.1 18.2±1.0 466.0 16
Antimicrobial Assays
17
Antibacterial assay
18
(Bauer et al., 1966)
Cont…
Sr. No. Sample. Code Enterobacter aerogens Escherichia coli Micrococcus luteus
Staphylococcus aureus
(mm)
1 A2 . - . 9.1±1.1
2 E2 . . . 18.5±0.3
3 A3 . . . 16.0±0.7
4 E3 . . . 8.3±0.9
5 A4 . . . 8.5±1.0
6 E4 . . . 8.3±1.1
7 A6 . . . 5.2±1.2
8 E6 9.6±0.9 . . 6.8±0.9
9 A7 . 17.6±0.6 . .
10 E7 . . . 6.9±1.0
11 A8 . . . .
12 E8 . . . .
13 A9 . . . .
14 E9 . . . .
15 A10 . . .
16 E10 . . . 8.7±0.7
17 A11 . . . .
18 E11 . . . .
19 A12 . . . .
20 A13 . . . .
21 E13 . . 11.6±1.1 . 19
22 Kanamycin 29.2 26.1 29.1 29.1
Micrococcus luteus
20
Escherichia coli
21
Staphylococcus aureus
22
Enterobacter aerogens
23
Antifungal assay
• Mucor species
• Fusarium solani
• Aspergillus flavus
• Aspergillus fumigates
• Aspergillus niger
24
(Esteban et al ., 2005)
Cont…
Aspergillus fumigates no activity
Aspergillus niger no activity
Fusarium solani no activity
Mucor species no activity
Aspergillus flavus activity
25
Aspergillus fumigates
26
Mucor species
27
Fusarium solani
28
Aspergillus niger
29
Aspergillus flavus
30
Brine Shrimp Cytotoxicity Assay
31
(Mclaughlin and Rogers 1998)
Cont…
• No cytotoxicity
32
Potato Disc Antitumor Assay
33
(Ahmad et al ., 2008)
Cont…
no activity
34
Cont…
Sr. No. Sample Percentage inhibition IC50 µg/ml
35
DNA-Drug Interaction Assay
36
(Arslantas et al., 2007)
Cont…
• spectrophotometer
• Addition of DNA in pure sample results in change in
absorbance or peak shift
• Hyperchromism
37
Cont…
2
Absorbance
1.5
0.5
0
230 280 330 380
Wavelength (nm)
38
1
0.9 0.5 e-6M DNA + extract
0.8
Absorbance
0.7 1 e-6M DNA + extract
0.6
0.5 e-5M DNA + extract
0.5
0.4 1e-5M DNA + extract
0.3
only extract
0.2
0.1
0
250 270 290 310 330 350 370 390 410 430 450
Wavelength (nm)
39
4
3.5
2.5
Absorbance
Only extract
2 0.5e-6M DNA + extract
1e-6M DNA + extract
1.5 0.5e-5M DNA + extract
1e-5M DNA + extract
1
0.5
0
320 370 420 470 520 570
Wavelength (nm)
1.2
1
Absorbance
Only extract
0.8
0.5e-6M DNA + extract
1e-6M DNA + extract
0.6
0.5e-5M DNA + extract
1e-5M DNA + extract
0.4
0.2
0
260 310 360 410 460
Wavelength (nm)
42
Acknowledgment
43
References
• Ahmad MS, Hussain M, Hanif M, Ali S, Qayyum M and Mirza B (2008). Di‐and
Triorganotin (IV) esters of 3, 4‐Methylenedioxyphenylpropenoic acid: synthesis,
spectroscopic characterization and biological screening for antimicrobial,
cytotoxic and antitumor activities. Chem Biol Drug des 71(6): 568-576.
• Arslantas A, Devrim AK and Necefoglu H (2007). The interaction of sheep
genomic DNA with a cobalt (II) complex containing p-nitrobenzoate and n,n`-
diethylnicotinamide ligands. Int J Mol Sci 8(12): 1225-1233.
• Bauer AW, Kirby WMM, Sherris JC and Turck M (1966). Antibiotic susceptibility
testing by a standardized single disk method. Am J Clin Pathol 45(4): 493-496.
• El-Said, SD (2012). Detection of parasites in commonly consumed raw
vegetables. Alexandria Med J 48(4): 345-352.
• Esteban A, Abarca ML and Cabanes FJ (2005). Comparison of disk diffusion
method and broth microdillution method for antifungal susceptibility testing of
dermatophytes. Med Mycol 43(1): 61-66.
44
Cont…
• Jabeen F, Oliferenko PV, Oliferenko AA, Pillai GG, Ansari FL, Hall CD and
Katritzky AR (2014). Dual inhibition of the α-glucosidase and butrylcholinesterase
studied by Molecular Field Topology Analysis. Eur J Med Chem 80: 228-242.
• Keerthana G, Kalaivani MK and Sumathy A (2013). In-vitro alpha amylase
inhibitory and anti-oxidant activities of ethanolic leaf extract of Croton
bonplandianum. Asian J Pharm Clin Res 6(4): 32-36.
• Koopman WJ, Guetta E, van de Wiel CC, Vosman B and van den Berg RG (1998).
Phylogenetic relationships among Lactuca (Asteraceae) species and related genera
based on ITS-1 DNA sequences. Am J Bot 85(11): 1517-1530.
• Matsui T, Tanaka T, Tamura S, Toshima A, Tamaya K, Miyata Y, Tanaka K and
Matsumoto K (2007). α-Glucosidase inhibitory profile of catechins and theaflavins.
J Agric Food Chem 55: 99-105.
• McLaughlin JL and Rogers LL (1998). The use of biological assays to evaluate
botanicals. Drug Inform J 32: 513-524.
• Ryder E (1979). Leafy salad vegetables. A VI Publishing Co, Westport, CT.
• Vries IM (1997). Origin and domestication of Lactuca sativa L. Genet Resour and
crop Ev 44(2): 165–164.
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